Amino acids are used in human medicine, in the pharmaceuticals industry, in the food industry and very especially in animal nutrition. It is known that amino acids are produced by fermenting strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to their great importance, the production processes are the subject of continuous improvement. Methodological improvements may relate to measures of fermentation technology such as, for example, stirring and oxygen supply, or the composition of the nutrient media such as, for example, the sugar concentration during fermentation, or the work-up to give the product form by, for example, ion exchange chromatography, or the intrinsic performance properties of the microorganism itself.
To improve the performance properties of these microorganisms one uses methods of mutagenesis, selection and mutant selection. In this manner, one obtains strains which are resistant to antimetabolites or which are auxotrophic for important regulatory metabolites and which produce amino acids. A known antimetabolite is the lysine analog S-(2-aminoethyl)-L-cysteine (AEC).
Methods of recombinant DNA technology for the strain improvement of L-lysine-producing strains of corynebacterium have also been employed for several years, by amplifying individual amino acid biosynthesis genes and studying the effect on amino acid production. The chromosome of Corynebacterium glutamicum has been sequenced completely a while ago (Kalinowski et al., Journal of Biotechnology 104:5-25 (2003)). The nucleotide sequence of the genome of Corynebacterium glutamicum R has been described in Yukawa et al. (Microbiology 153(4):1042-1058 (2007)). The chromosome of Corynebacterium efficiens has likewise already been sequenced (Nishio et al., Genome Res. 13 (7):1572-1579 (2003)). The relevant sequence information can be found in the public databases. Suitable databases are, for example, the database of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany, and Cambridge, UK), the database of the National Center for Biotechnology Information (NCBI, Bethesda, Md., USA), that of the Swiss Institute of Bioinformatics (Swissprot, Geneva, Switzerland), the Protein Information Resource Database (PIR, Washington, D.C., USA) and the DNA Data Bank of Japan (DDBJ, 1111 Yata, Mishima, 411-8540, Japan).
Summarizing reviews of the genetics, the metabolism and the technical importance of corynebacterium are found in the papers of Ikeda, of Pfefferle et al. and of Mueller and Huebner in the book “Microbial Production of L-Amino Acids” (Advances in Biochemical Engineering 79 (2003), Springer Verlag, Berlin, Germany, editor: T. Scheper), in the special edition “A New Era in Corynebacterium glutamicum Biotechnology” of the Journal of Biotechnology (volume 104 (1-3), 2003, editor: A. Pühler and T. Tauch) and in the “Handbook of Corynebacterium glutamicum” (editor: L. Eggeling and M. Bott, CRC Press, Taylor & Francis Group, Boca Raton, Fla., USA, 2005).
The nucleotide sequence of the dapB gene which codes for the Corynebacterium glutamicum dihydrodipicolinate reductase is publicly available, inter alia, in the database of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA) under the accession number NC—006958 (region: 2051238-2051984 (complementary)) including the upstream and downstream regions. It can furthermore be found in the patent application WO 0100843-A. DapB catalyzes the reduction of 2,3-dihydrodipicolinate to 2,3,4,5-tetrahydrodipicolinate in lysine and diaminopimelate biosynthesis. According to Cremer et al. (Applied and Environmental Microbiology, 57(6): 1746-1752 (1991)), the overexpression of dihydrodipicolinate reductase alone does not improve the secretion/excretion of L-lysine.
Gene expression is controlled, inter alia, by the promoter region in the 5′ region of a gene. Transcription initiation takes place in the promoter as the result of the interplay between transcription factors and RNA polymerase. This is why a series of conserved sequence motifs are present in promoters which can also be determined in Corynebacterium glutamicum (Patek et al., Microbiology 142: 1297-1309 (1996)) analogously to the general bacterial promoter elements classified in the best-studied bacterial model organism Escherichia coli analogously to the genes transcribed with the aid of the sigma-70 factor (Rosenberg et al., Nature 272:414-423 (1978); Hawley and McClure, Nucleic Acids Research 11(8):2237-2255 (1983); Fournier et al., Antimicrobial Agents and Chemotherapy 39(6):1365-1368 (1995); Chapon, EMBO Journal 1:369-374 (1982); Smith et al., Journal of Bacteriological Chemistry 257:9043-9048 (1982)):                the −35 region (the sequence located 35 base pairs upstream of the transcription start), with the consensus sequence: 5′-tttGcca.a-3′,        the −10 region (this sequence is located approximately 10 base pairs upstream of the transcription start), also referred to as Pribnow box, with the consensus sequence: 5′-ggTA.aaT-3′.        
The sigma factor of the RNA polymerase which then initiates the transcription of the downstream gene/ORF binds to these two regions. So-called consensus sequences for strong and weak promoters can be deduced from the comparison of the DNA sequences of individual promoters.
The position of the promoter elements relative to one another and/or to the transcription start is of importance, too. The distance of the −10 region to the transcription start is five to seven base pairs in the consensus sequence, the −10 region and the −35 region are 16 to 18 base pairs apart.
The similarity of a promoter with the consensus sequence decides the transcription rate of a gene and thus contributes to the expression level. In Corynebacterium glutamicum, the −35 region is markedly less conserved than the −10 region.
If mutations are performed in the regulatory sequence upstream of the start codon, the functionality of these elements as a function of the sequence and of the distances to the start codon must be taken into consideration.
For reasons of clarity, the nucleotide sequence of the coding region (CDS) of the dapB gene coding for the dihydrodipicolinate reductase of Corynebacterium glutamicum wild type (“wild-type gene”) is shown in SEQ ID NO:1 in accordance with the specifications of the NCBI database and the resulting amino acid sequence of the encoded dihydrodipicolinate reductase is shown in SEQ ID NO:2 and 4. SEQ ID NO:3 additionally shows nucleotide sequences (in each case approximately 1000 nucleotides) which are located upstream and downstream of the CDS.